Immunomodulation.
Many disease conditions are, at least in part, a result of an unwanted or excessive immune response within an organism. The rejection of a transplanted organ is axiomatic example of an unwanted immune response. The rejection of the graft is emblematic of a condition in which an organism's inability to control an immune response results in a pathology. In organ transplantation, the unwanted immune response that results in graft rejection is triggered by: (1) “direct recognition,” where the T cells of the graft recipient recognize foreign major histocompatibility complex (“MHC”) molecules on the graft tissue, already presenting some peptides, via their T-cell receptor (“TCR”) directly, or “indirect recognition,” where the recipient T cells recognize the antigenic determinants derived from the graft after the determinants are processed and presented by recipient MHC; (2) the generation of antibodies directed against the graft, more specifically, the human leukocyte antigens (“HLA”) molecules present on the cells of the graft tissue, caused by the exposure of the recipient to the graft; and (3) binding of preformed anti-graft antibodies in the circulation of the recipient to the graft. Studies have shown that these immune responses are directed to three types of donor derived antigens: MHC (through direct or indirect recognition), minor histocompatibility antigens (“mH”), and organ derived antigens.
Successful transplantation depends on preventing the unwanted immune responses, inducing sustained chimerism. Sustained chimerism is a phenomenon in which the recipient develops tolerance for a foreign graft, enabling the grafted tissue to survive in the recipient without being subjected to immune responses. Under experimental conditions, sustained chimerism can be induced by peptides that are closely related to those that stimulate graft-rejecting immune responses, albeit for short periods of time. (B. Murphy et al., J. Am. Soc. Nephrol., 2003, 14:1053-1065; C. LeGuern, Trends Immunol., 2003, 24:633-638). The difficulty lies with the likelihood of the broadening of the offending epitopes via the process of epitope spreading (N. Suciu-Foca et al., Immunol. Rev., 1998, 164:241).
Transplant physicians have long recognized the need both to inhibit the immune response generated by the presence of what the recipient's immune system views as foreign, without also compromising the patient's ability to fight opportunistic infection. Currently, transplantation patients are often treated with immunosuppressive therapies that depress the overall immune response and reactivity in a patient. Immunosuppressive therapies attempt to attenuate the reaction of the body to an already-triggered immune response, and are accompanied by numerous undesirable side effects. Because of the significant undesirable side effects, a single immunosuppressant cannot be used continuously to treat a transplant recipient, and a course of treatment comprises using one immunosuppressant having one set of side effect, changing to second immunosuppressant with a different set of side effect, and to third, and so on, to limit the exposure of the recipient to each immunosuppressant and its side effects. For example, steroids such as prednisone or methylprednisone are powerful immunosuppressants but can induce cataracts, hyperglycemia, hirstutism, bruising, acne, bone growth suppression, and ulcerative oesophagitis. Long term use of steroids has also been associated with bone loss. Cyclosporin A (CsA), a widely used immunosuppressant, is nephrotoxic, and often replaced with tacrolimus (TAC) after a period of treatment. For the treatment of non-acute rejection, azathioprine is used, the side effect of which include leucopenia, anemia, fever, chills, nausea and vomiting. Regardless of what immunosuppressant is used, one of the most substantial side effects related to longer term treatment with immunosuppressives in addition to the general compromise of the immune system leaving the patient vulnerable to any type of infections, is the generation of transplant related malignancies such as Kaposi's sarcoma. There is a strong desire on the part of physician and patient to decrease or cease the use of these current front line therapies. (Pharmacotherapy: A pathophysiologic Approach, Fifth Edition. 2002, McGraw Hill.) It would be difficult to state that they have met the clinical goal of sustained chimerism without ongoing immunosuppressive therapy.
Immunomodulation, in contrast to immunosuppression, targets the cause of unwanted immune responses. Immunomodulation can be attempted in an antigen/epitope non-specific fashion by targeting the body's mechanism for immunity, or in an antigen/epitope specific manner. As an example of antigen/epitope non-specific treatment, therapies directly targeted at controlling T lymphocytes or their functions have been developed using biotechnological tools. The therapeutic agents useful for such treatment include Muromonab-CD3 (OKT3), antilymphocyte globulin (ALG), antithymocyte globulin (ATG), or interleukin-2 receptor monoclonal antibody (“mAb”) daclizumab or basiliximab. Other agents include soluble CTLA-4, an anti-CD154 mAb; anti-CD11a; a humanized mAb which inhibits VLA-4; anti-CD2, 3, or 4 antibodies; and anti-CD152 antibodies (J. B. Matthews et al., Amer. J. Transplantation, 2003, 3: 794-80). While all of these therapeutic agents may induce a state of non-responsiveness of the recipient's immune system to the transplanted tissue with a reduction in side effects, as compared to e.g. prednisone, the therapies still do not meet the clinical goal of sustained chimerism without ongoing immunosuppressive therapy, except for limited reports, such as immunosuppressive withdrawal after combination therapy of total lymphoid irradiation followed by ATG administration (S. Strober et al., Transplantation, 2004 Mar. 27; 77(6): 932-936). Further, these therapies also suffer from the unattractive side effects of compromised overall immune function.
In contrast to the antigen non-specific immunomodulatory approach, the immune system can also be retuned, or modulated in an antigen/epitope specific manner. Such a type of immunomodulation is the process of increasing or decreasing the immune system's ability to mount a response against a particular antigenic determinant through either the TCR's recognition of complexes formed by MHC and antigens, or through the B cell receptor's (“BCR”) recognition of the epitope itself. Because of the specificity of the process toward a particular antigenic determinant and not toward the immune system as a whole, antigen specific immunomodulation has advantages such as fewer undesirable side effects compared to current treatment modalities such as immunosuppressive therapies, which affects the overall immune system.
Antigenic determinant-specific immunomodulatory treatments can help establish such sustained chimerism by inducing donor-specific tolerance in host T lymphocytes. Immunomodulation of the reaction toward any and all of these antigens help attenuate or alleviate graft rejection and establish sustained chimerism. Studies indicate that one mechanism of action of immunomodulation by certain immunomodulatory peptides may be through their binding to T cells that would otherwise bind to the donor-derived antigens and resulting in differential activation of T cell functions. This mechanism has been suggested to be centrally induced tolerance involving the thymus (G. Benichou et al. Immunol. Today, 1997, 18(2):67-72). The demonstration of achieving sustained chimerism without immunosuppressive treatment via induction of donor-specific tolerance in host T lymphocytes through immunomodulation was performed by a group of investigators who, using mice, induced tolerance to the subsequent graft by intrathymic injection of a series of determinants from 3M KCl-extracted donor MHC-derived peptides. Two doses of anti-T cell antibody were given first to eliminate circulating T cells. Then eight peptide sequences extracted from the donor MHC were delivered in combination. The treated mice tolerated subsequent transplants. As a control, the investigators performed thymectomy, which caused graft rejection. The study is an example of importance of centrally-induced tolerance (T. Hamashima et al., Transplantation, 1994 Jul. 15; 58(1):105-7). Thus, designing appropriate peptides similar to T cell-stimulating antigens that bind to the T cells is beneficial to achieving sustained chimerism.
However, the difficulty lies with the likelihood of the broadening of the offending epitopes via the process of epitope spreading. (N. Suciu-Foca et al., Immunol. Rev. 1998, 164:241). Thus, in transplantation, the axiomatic example where certain immune response is unwanted, it is clear that, in the absence of the ability to modulate the relevant antigenic determinants over time, the only alternatives are non-specific immunomodulatory, or immunosuppressive therapies.
Other examples of unwanted immune responses are autoimmune diseases. One important contextual difference between autoimmune diseases and transplantation rejection is that the offending antigenic determinant(s) is/are generally more restricted and definable. While the trigger of an autoimmune disease is undefined and may be dictated by pre-existing and/or environmental factors, the direct causes of the pathological condition have been identified in many autoimmune diseases. An autoimmune disease results from an inappropriate immune response directed against a self antigen (an autoantigen), which is a deviation from the normal state of self-tolerance. Self-tolerance arises when the generation of T cells and B cells capable of reacting against autoantigens has been prevented or altered centrally by events that occur either in their early development or after maturation in the periphery. The cell surface proteins that play a central role in regulation of immune responses through their ability to bind and present processed peptides to T cells via the T cell receptor (TCR) are class I and class II MHC (J. B. Rothbard et al., Annu. Rev. Immunol., 1991, 9:527).
Thus, an attractive point of intervention for the amelioration of an autoimmune response is via the set of lymphocyte surface protein MHC molecules for example, HLA-DR, -DQ and -DP, themselves or in combination with the peptides they present. Different HLA alleles generate a diversity of responses via antigenic-determinant specificities by variable affinities for protein fragments found in the extra- and intra-cellular milieu because of differences in the amino acids which are directly involved in the binding of the peptides. There are large numbers of alternative or allelic forms within a mammalian population, but only a few of these allelic forms are associated with disease-related antigenic determinants. It is well understood to one with ordinary skill in the art the genomes of subjects affected with certain autoimmune diseases, for example MS and RA, are more likely to carry one or more such characteristic MHC class II alleles, to which that disease is linked. For example, HLA-DR2 (DRB1*1501) is associated with multiple sclerosis and HLA-DR1 (DRBI*0101) or HLA-DR4 (DRB1*0401) are associated with rheumatoid arthritis.
The disease-related antigenic determinants derive from proteins which have been described as being simply associated with an autoimmune response, or as being part of the pathogenesis of the disease process itself. There are highly conserved sequences within HLA that may play a role in either the generation or regulation of immunologic tolerance when processed into peptides and presented by intact HLA (reviewed in B. Murphy and A. M. Krensky, J. Am. Soc. Nephroi., 1999, 10:1346-55). A. Snijders et al. discuss one particular sequence (KDILEDERAAVDTYC) (SEQ ID NO: 206) presented by HLA-DRB1 as being protective against rheumatoid arthritis, with the most relevant portion of the peptide being DERAA (SEQ ID NO: 207) (J. Immunol., 2001, 166:4987-93), while others have promoted what is known as the ‘shared epitope hypothesis’ (P. K. Gregersen et al., Arthritis Rheumatism 1987 November; 30(11):1205-13) where those individuals that carry HLA-DRB1 alleles having the sequence QKRAA (SEQ ID NO: 208) are predisposed to rheumatoid arthritis. Other investigators have demonstrated that heat shock proteins (hsp) and the peptides derived from them can have immunomodulatory properties (S. M. Anderton et al., J. Exp. Med., 1995, 181:943-952; J. A. van Roon et al., J. Clin. Invest., 1997, 100:459-063). One peptide in particular, dubbed p277, derives from hsp60, VLGGGVALLRVIPALDSLTPANED (SEQ ID NO: 147), has demonstrated apparent activity in the context of Type I diabetes (I. Raz et al., Lancet, 2001, 358:1749-52). Further sources of epitope sequence may be derived from a pathogen-derived mimic of a sequence within mammalian MHC proteins such as the DNAjP1 peptide, or related peptides (QKRAAYDQYGHAAFE (SEQ ID NO: 209); Proc. Nat. Acad. Sci. USA, 101:4228-33; U.S. Pat. No. 6,989,146). Other proteins and the peptides that derive from them having disease association are: glutamate decarboxylase (GAD) with diabetes (M. A. Atkinson et al. J. Clin. Invest., 1994, 94:2125-29; D. B. Wilson J. Autoimmun., 2003, 20:199-201); myelin associated proteins such as myelin basic protein (MBP), myelin-associated glycoprotein (MAG), proteolipid protein (PLP), and myelin oligodendrite glycoprotein (MOG) with multiple sclerosis (reviewed in P. Fontoura et al., Int. Rev. Immunol., 2005, 24:415-46); Ro60, SmD and other ribonucleoprotein antigens with lupus (R. Pal, et al., J. Immunol., 2005, 175:7669-77; Seshmukh et al., J. Immunol., 2000, 164:6655-61; R. R. Singh, Mol. Immunol., 2004, 40:1137-45); or the acetylcholine receptor (AChR) with myasthenia gravis (MG) (S. L. Kirshner, et al. Scand. J. Immunol., 1996, 44:512-21); or desmoglein 3 (DsG3) with pemphigus vulgaris (PV) (Wucherpfennig et al., Proc. Nat. Acad. Sci. USA, 1995, 92:11935-9; Lin et al., J. Clin. Invest., 1997, 99:31-40; Veldman et al., J. Immunol., 2004 172:3883-92; Angelini et al., J. Translational Med., 2006, 4:43; U.S. Pat. No. 5,874,531; U.S. Pat. No. 7,084,247).
Despite the attraction of using HLA alleles and their associated antigenic determinants that have been linked to many autoimmune diseases as a point of intervention, therapeutic agents based on this knowledge have not been developed fully. Instead, a number of immunomodulatory therapeutic agents that are not specific to any particular antigenic determinant have been developed and being used to treat autoimmune diseases, including general anti-inflammatory drugs such as cyclooxygenase-2 (COX-2) inhibitors that can prevent formation of low molecular weight inflammatory compounds; inhibitors of a protein mediator of inflammation such as tumor necrosis factor (TNF), such as an anti-TNF specific monoclonal antibody or antibody fragment, or a soluble form of the TNF receptor that sequester TNF; and agents that target a protein on the surface of a T cell and generally prevent interaction with an antigen presenting cell (APC), for example by inhibiting the CD4 receptor or the cell adhesion receptor ICAM-1. However, these types of antigenic-determinant non-specific immunomodulatory therapeutic agents have residual immunosuppressive-like side-effects which diminish their attractiveness as chronic first line therapies. Additionally, compositions having natural folded proteins (such as antibodies) as therapeutic agents can encounter problems in production, formulation, storage, and delivery. Several of these problems necessitate delivery to the patient in a hospital setting.
Strategy for Creating Synthetic Therapeutic Peptides
Drug discovery can be generalized into two major elements, lead generation and lead optimization. The development and exploitation of combinatorial chemistry (CC) has seen the divergence of the uses of rational design versus random generation on a very fundamental level. On one side we find the use of CC to assist a researcher in the rational design of molecules. An example of which can be seen in the discovery of structure/activity relationships (SAR) between two or more active molecules of therapeutic interest. On the other side we find researchers using CC to define for them the design of new molecules discovered based on a specific activity. An example of which would be the generation of random libraries used in lead generation, whereby the lead is singled out and further optimized.
The level of expertise in the state of the art of combinatorial chemistry as applied to the synthesis of peptide libraries has risen, producing highly reliable and pure mixtures of peptides of great diversity. The use of these diverse peptide libraries has focused on lead generation and optimization. This strategy entails screening the vast numbers of individual peptide sequences in the library against a target of interest with the intention of defining a single, or limited set of peptides which demonstrate a particular activity. That single peptide, or the limited set of peptides, then become candidates which are modified to increase activity against the target. This process is schematically represented in FIG. 1A.
The challenge for practitioners in this art has been to deconvolute, or accurately define the single or limited set of peptides that were responsible for the observed activity. The difficulties associated with deconvolution have spawned great efforts on the part of practitioners to create synthesis methods which inherently increase the resolution of individual peptides, as well as the identity of individual amino acids within peptides.
In order to efficiently identify the target peptide from myriad of candidates presented by a library created by combinatorial chemistry, a variety of synthesis methods and approaches have been developed. These synthesis methods aim to provide a large number of candidates, and yet when a positive result is obtained, to quickly determine the identity of the peptide without having to laboriously isolate the positive species from the rest. The effort put forth by practitioners in this art in this regard is an indication of the industry-wide vision of the method's ultimate utility, which is to allow the random complexity of these libraries perform the screening process for the desired activity.
Examples of the resulting evolution of subtypes of combinatorial methods include: multiple synthesis, iterative synthesis, positional scanning, and one-compound-one-bead post assay identification design.
Multiple synthesis” provides for any method whereby distinct compounds are synthesized simultaneously to create a library of isolated compounds. The identity of these compounds would be known from the rules of the synthesis. H. M. Greysen et al., Proc. Nat. Acad. Sci. USA, 1984, 81:3998, used the multiple synthesis method to identify peptides that bound to an antibody raised against VPI protein of foot-and-mouth disease virus. The investigators identified GDLQVL (SEQ ID NO: 210) as the epitope recognized by the antibody. In this case the authors synthesized 108 overlapping peptides representing the VPI sequence on pins in a 96-well microplate array.
“Iterative synthesis/screening” involves methods of peptide synthesis which allow for a determination of the identity of individual residues within peptide sequences. An example of iterative synthesis can be seen in R. A. Houghten et al., Nature, 1991, 354:84-86, also to determine antibody binding epitopes. These investigators identified the sequence YPYDVPDYASLRS (SEQ ID NO: 211) using an ELISA type assay format. The first library consisted of 324 pools of peptides with the first two residues fixed, which peptides can be shown as O1O2XXXX, wherein O1 and O2 are the fixed residues and X is randomly selected. The process identified DV as the fix residues. The next step was to do the same for position three, by synthesizing peptides that can be shown as DV O1XXX, wherein O1 again is a fixed residue. When the process identified which residue at the third position would elicit the desired binding, that residue was adopted as the unchanging third residue, and the fourth position was explored in a similar manner. The process continued until the native sequence DVPDYA (SEQ ID NO: 212) was identified.
“Positional scanning” is a synthesis method producing complex mixtures of peptides that allows for the determination of the activity of each individual peptide. Based on the screening results, the derived peptide can then be separately synthesized for optimization. As seen in C. Pinilla et al., Biochem J., 1994, 301:847-853, positional scanning libraries were used to identify decapeptides which bound the same YPYDVPDYASLRS-binding (SEQ ID NO: 211) antibody. In this case ten different libraries each containing 20 pools with a defined amino acid at each of the ten positions in the peptide. Fifteen peptides were identified.
Each of the above methods were also employed to identify enzyme substrates (J. H. Till et al., J. Biol. Chem., 1994, 269:7423-7428, J. Wu et al, Biochemistry, 1994, 33:14825-14833, W. Tegge et al., Biochemistry, 1995, 34:10569-10577), or enzyme inhibitors (M. Bastos et al., Proc. Nat. Acad. Sci. USA, 1995, 92:6738-6742, Meldal et al., Proc. Nat. Acad. Sci. USA, 1994, 91:3314-3318), R. A. Owens et al., Biomed Biophys. Res. Commun., 1994, 181:402-408, J. Eichler. et al., Pept. Res., 1994, 7:300-7). These powerful tools allow investigators to rationally design combinatorial peptide libraries to identify a single species which has a desired activity.
As powerful and clear cut the identification of a specific peptide from a combinatorial library may be, it may only serve as a starting point and identification of a lead peptide that is not itself therapeutically useful. The identified epitope may be ignored by the immune system if it resembles a self protein or possibly exacerbate the very condition that the therapy aims to relieve. Such peptide is not directly therapeutically useful. However, one may create, based on such peptide, epitope reactive analogs that would act as modifiers of the unwanted immune response.
One such approach is creation of altered peptide ligands (APL). This approach is schematically represented in FIG. 1B. An APL is defined as an analog peptide which contains a small number of amino acid changes from the native immunogenic peptide ligand. Some of such APLs act as an antagonist to the T cell receptor, blocking the stimulating binding by the antigens causing the unwanted immune effect. Evabold et al., Proc. Nat. Acad. Sci. USA, 1994 Mar. 15; 91(6):2300-4. However, while recognition of the native response may induce an angonist like reaction, an APL might induce a partial agonist response, or induce a state of energy in the reactive T cell population. In discussing APL in the context of allograft rejection therapy, Fairchild et al., Curr. Topics Peptide Protein Res., 2004, 6:237-44, note that an APL acting as an antagonist for one TCR, may become an agonist for another, complicating the rational design of an APL. Compounding the obstacle of the development of APL is the difficulty in translating a response developed in an animal system into human.
Despite these challenges, MPB83-99 (ENPVVHEFKNIVTPRTP) (SEQ ID NO: 213) was made into an APL and placed into limited human trials by replacing the bold and underlined amino acid residues “E”, “N”, “E” and “K,” resulting in a single peptide sequence consisting of AKPVVHLFANIVTPRTP (SEQ ID NO: 214), Kim et al. Clinical Immunology, 2002, 104:105-114. The authors describe the long term immune reactivity against the peptide, but the treatment has been deemed clinically ineffective by evaluation using MRI. Thus an APL, once identified, can be used as a therapeutic agent; however, its effectiveness may be limited in terms of clinical efficacy.
It has been observed for some time that in the course of development of multiple sclerosis, the reactive epitope does not stay constant. That is, the self recognition associated with the development of MS is a developmental process characterized by autoreactive diversity, plasticity, and instability, wherein the target epitope changes over time, typically from one epitope on a myelin proteolipid protein to one overlapping the amino acid residues but shifting by one or few amino acids to either side of the original epitope. The consequence of this phenomenon is that if an immunotherapeutic drug was targeted at the original epitope, over time, it becomes ineffective, not because of resistance to the mechanism of the drug, but simply because the target is no longer valid. J. Clin. Invest., 1997, 99:1682-1690.
A method conceived to make an investigational concept like a mixture of peptides into a drug is peptide dendrimer structures. Peptide dendrimers solve certain manufacturing issue of soluble peptide mixtures, in part by the promise of delivering to a patient a consistent ratio and quantity of each of the peptides in the mixture. This approach is schematically represented in FIG. 1C.
Dendrimers are diverse. They can range in size from 2 kDa to greater than 100 kDa. The design of dendrimers intends to mimic two traits of naturally occurring biological structures: a globular structure and polyvalency. As described in two comprehensive reviews (P. Niederhafier et al., J Peptide Sci. 11:757-788; K. Sadler and J. P. Tam, Rev. Mol. Biotechnol., 2002, 90:195-229), they are complex compounds that contain highly branched components organized in a radial or wedge-like fashion, and are intended to have an extensive three-dimensional structure. They have three distinct structural features: a central core surface functionalities and branching units that link the two. Peptide dendrimers are designed as vehicles for delivery of: RNA and DNA as gene expression therapeutics, biosensor systems as diagnostics, inhibitors of autoimmune diseases or cancer metastasis. The strategy behind each of these applications is to use the globular, polyvalent structure to amplify the ligand:substrate interaction (D. Zanini and R. Roy, J. Org. Chem., 1998, 63:3468-3491; J. Haensler and F. C. Szoka, Bioconjug Chem., 1993, 4:372-379).
Dendrimers have been made using amino, hydroxyl, carboxy, poly(propylenimine), silicone and polyamino amine cores (G. M. Dykes et al., J. Chem. Technol. Biotechnol., 2001, 76:903-918, P. Sadler and J. Jezek, Rev. Mol. Biotechnol., 2002, 80:195-229, and J. P. Tam, Methods Org. Chemistry, 2004, Vol E22d 129-168. Peptide dendrimers can be divided into three types: grafted peptide dendrimers, branching polyamino acids and multiple antigen peptides (MAPs).
The branching strategies in MAPs vary widely. The majority of first generation branches have used lysine. Second generation solid phase synthesis of MAPs has seen an interest in proline. The interest is said to come from both the properties of its secondary amine which decreases the reactivity during production, as well as its role in many cellular functions.
Simple MAPs have been synthesized using solid phase chemistry, with this type of synthesis strategy called divergent. Synthesis methods have been described which involves a two-step iterative reaction sequence producing concentric shells of dendritic beta-alanine units covalently linked in the second step to various functional groups (Kojima et al., Bioconjugate Chem., 2000, 11:910-17). These types of MAPs, which are synthesized using the divergent strategy, by necessity have simple branching schemes with few distinct members, as the purification and characterization are untenable with more complex MAPs. The end-product needs to be purified away from deletion compounds having similar characteristics to the end-product. Purifications have been described using gel filtration chromatography, reverse phase high-performance liquid chromatography (HPLC), or electromigration methods.
For complex MAPs, for example, those having a multiplicity of branching moieties, convergent synthesis is the preferred synthesis strategy. Convergent synthesis can be performed using either fragment condensation or ligation of the pre-purified fragments. There are many types of ligations: natural (true peptide bond created), thiol, hydrazone, or other. MAPs prepared using convergent synthesis strategies are easier to purify, as the end-product will look distinctly different from the reaction byproducts. HPLC was first used to purify convergent MAPs (J. C. Spetzler et al., Int. J. Pept. Protein Res., 1995, 45:78-85).
However, a high cost of manufacturing and the subsequent analytical development precludes this technology from being further currently developed commercially.
All of the above strategies, while recognizing the advantage of variations in the therapeutic peptide compositions, derive from the concept that there is one or more defined peptide sequence evoking a defined immunological response. These strategies have attempted to multiply and diversify modulatory peptides via the introduction of defined, single changes performed one at a time.
An entirely different approach which has evolved alongside the defined sequence peptide immunotherapy approach is the use of limited amino acid diversity, random epitope polymers. Random sequence polymers (RSP) can be described as a random order mixture of amino acid copolymers comprising two or more amino acid residues in various ratios, forming copolymers by random sequence bonding, preferably through peptide bonds, of these amino acid residues, which mixture is useful for invoking or attenuating certain immunological reactions when administered to a mammal. Because of the extensive diversity of the sequence mixture, a large number of therapeutically effective peptide sequences are likely included in the mixture. In addition, because of the additional peptides which may at any given time not be therapeutically effective, but may emerge as effective as the epitope shifting and spreading occurs, the therapeutic composition may remain effective over a time of dosing regimen. This approach is schematically represented in FIG. 1D.
Starting in 1959 (P. H. Maurer et al., J. Immunol., 1959, 83:193-7) to 1988, (J. L. Grun, and P. H. Maurer, Immunogenetics, 1988, 28(1): 61-3) Maurer and colleagues investigated the immune responses to poly glutamic acid and other random sequence polymers such as those consisting of tyrosine, glutamate and alanine (YEA), phenylalanine, glutamate and alanine (FEA), and phenylalanine, glutamate and lysine (FEK). Teitelbaum et al., Eur. J. Immunol., 1971, 1:242-8 was the initial report of work on random copolymer consisting of tyrosine, glutamate, alanine and lysine, that eventually culminated in an FDA approved therapy for multiple sclerosis using COP-1, described below. In 1978, Germain and Benacerraf, J. Exp. Medicine 148:1324-37, investigated suppressor T cell responses to YEA in what was to become Benacerrafs 1980 Nobel winning work on the role of MHC in the immune system and its relevance to alloreactivity (http://nobelprize.org/nobel_prizes/medicine/laureates/1980/benacerraf-lecture.html).
Copolymer-1 (also known as Copaxone, glatiramer acetate, COP-1, or YEAK (SEQ ID NO: 217) random copolymer), is used for the treatment of multiple sclerosis. Random copolymers are described in International PCT Publication Nos. WO 00/05250, WO 00/05249; WO 0/59143, WO 0027417, WO 96/32119, WO/2005/085323, in U.S. Patent Publication Nos. 2004/003888, 2002/005546, 2003/0004099, 2003/0064915 and 2002/0037848, in U.S. Pat. Nos. 6,514,938, 5,800,808 and 5,858,964.